The present invention relates to separation of fluids, with subsequent analysis and evaluation. The fluid may be serum (plasma) lipid and may be separated by, liquid chromatography or electrophoresis, including capillary electrophoresis to determine the concentrations of components of the samples. Determination of concentration of components may be obtained with a densitometer. One particular, but non-limiting aspect of the present invention is cholesterol-triglyceride separation and subsequent analysis.
In the separation and analysis of serum lipid, lipid metabolism is analyzed by separation with respect to specific gravity, which is now an important examination for the treatment of hyperlipemia Serum lipid, when separated with respect to specific gravity by a centrifuge, can be separated into HDL (high-density lipoprotein), LDL (low-density lipoprotein, IDL (intermediate density lipoprotein), VLDL (very low-density lipoprotein), and CM (chylomicron). Since the separation is carried out by an ultracentrifuge, for finer and detailed separation to grasp conditions during separation, this method is difficult to be employed in-the clinical area because it requires much labor and time. Since separation of serum lipid by electrophoresis shows a good correlation with that of an ultracentrifuge and the separation condition can be visually obtained, the electrophoretic lipoprotein separation is employed in routine examination.
In the basic structure of lipoprotein, a core part is formed of triglyceride and cholesterol ester, which is covered with a single layer of film composed of phospholipid and free cholesterol, and a single to several types of apoprotein are attached to the surface. That is, cholesterol and triglyceride and phospholipid of α(HDL) of lipoprotein fraction, pre β(VLDL), between pre and β(IDL), β(LDL), and chylomicron part can be measured to determine ratios of components in the respective fractions. Since metabolism of lipid relative to enzyme and cholesterol transfer protein, and lipoprotein is slightly changed in particle size with the content of triglyceride, close examination of these conditions serves to detect an abnormality of metabolism.
In electrophoresis, lipid is separated into a position a (HDL), pre β position (VLDL), β(LDL), sample application position (chylomicron), and between pre β and β(IDL), and dyed with fat red 7B and the like for measurement. WHO classifies hyperlipemia into six phenotypes of I, IIa, IIb, III, IV, and V. However, in the prior art method where this classification is performed for lipoprotein fraction, total cholesterol value, total triglyceride value, and presence of chylomicron, by visual examination of a serum sample kept in a refrigerator for 12 hours, there have been problems in that the method takes much time, some samples are very difficult to evaluate, since visual examination calls for judgment, and classification is difficult even for skilled persons.
Phenotype classification of hyperlipemia is performed for pre β(VLDL+IDL), β(LDL) and change in chylomicron content, and pre β(VLDL+IDL) is mainly of triglyceride rich, whereas (LDL) is cholesterol rich. Chylomicron, since most of it is triglyceride, in general the phenotype of hyperlipemia is determined from the total triglyceride value (described in fraction name of ultracentrifugal method). That is, VLDL+IDL is determined from the total triglyceride value, and LDL from the total cholesterol value. The presence of chylomicron is determined by visual examination of serum stored in a refrigerator for 12 hours. In the WHO classification, the determination is made as follows.
a. In type I, VLDL+IDL and LDL are normal, whereas only chylomicron is-high.
b. In type IIa, only LDL is high, whereas VLDL+IDL and chylomicron are normal.
c. In type IIb, VLDL+IDL and LDL are high, whereas chylomicron is normal.
d. In type III, VLDL+IDL and LDL approach each other to form broad E in the lipoprotein fraction, and determination is impossible without lipoprotein fractionation.
e. In type IV, VLDL+IDL is high, whereas LDL and chylomicron are normal.
f. In type V, chylomicron and VLDL+IDL are high, whereas LDL is normal. To perform such classification, in general, a visual judgment is made with reference to a guideline for hyperlipemia of the Society of Arteriosclerosis. However, since, with the above-described method, lipoprotein fraction can be determined only in ratio, and chylomicron is qualitatively analyzed, it is difficult to make judgment of phenotype automatically. Further, in some analytes, VLDL+IDL contains a large amount of cholesterol, LDL contains a large amount of triglyceride, and such abnormal analytes cannot be detected by the above classification.